Primary Acute Myelogenous Leukemia Research Articles

Therapeutic Targeting of TIM-4-L with Engineered T Cells for Acute Myeloid Leukemia.

Disruption of lipid bilayer asymmetry is a common feature observed in cancer cells and offers novel routes for therapeutic targeting. We used the natural immune receptor TIM-4 to interrogate for loss of plasma membrane phospholipid polarity in primary acute myelogenous leukemia (AML) samples and evaluated the anti-leukemic activity of TIM-4-L-directed T-cell therapy in preclinical AML models. We performed FACS analysis on 33 primary AML bone marrow specimens and correlated TIM-4-L expression frequency and intensity with molecular disease characteristics. Using Kasumi-1 and MV-4-11 AML cell lines, we further tested the anti-leukemic effects of TIM-4-L-directed engineered T cells in vitro and in vivo. We found that 86% of untreated AML blasts displayed upregulation of cell surface TIM-4-L. These observations were agnostic to AML genetic classification, as samples with mutations in TP53, ASXL1, and RUNX1 displayed TIM-4-L upregulation similar to that seen in favorable and intermediate subtypes. TIM-4-L dysregulation was also stably present in AML cell lines. To evaluate the potential of targeting upregulated TIM-4-L with adoptive T-cell therapy, we constructed TIM-4-L-directed engineered T cells, which demonstrated potent anti-leukemic effects, effectively eliminating AML cell lines with a range of endogenous TIM-4-L expression levels both in vitro and in vivo. These results highlight TIM-4-L as a highly prevalent target on AML across a range of genetic classifications and novel target for T-cell-based therapy in AML. Further investigations into the role of TIM-4-L in AML pathogenesis and its potential as an anti-leukemic target for clinical development are warranted.

Human acute leukemia uses branched-chain amino acid catabolism to maintain stemness through regulating PRC2 function.

Cancer-specific metabolic activities play a crucial role in the pathogenesis of human malignancies. To investigate human acute leukemia-specific metabolic properties, we comprehensively measured the cellular metabolites within the CD34+ fraction of normal hematopoietic stem progenitor cells (HSPCs), primary human acute myelogenous leukemia (AML), and acute lymphoblastic leukemia (ALL) cells. Here, we show that human leukemia cells are addicted to the branched-chain amino acid (BCAA) metabolism to maintain their stemness, irrespective of myeloid or lymphoid types. Human primary acute leukemias had BCAA transporters for BCAA uptake, cellular BCAA, α-ketoglutarate (α-KG), and cytoplasmic BCAA transaminase-1 (BCAT1) at significantly higher levels than control HSPCs. Isotope-tracing experiments showed that in primary leukemia cells, BCAT1 actively catabolizes BCAA using α-KG into branched-chain α-ketoacids, whose metabolic processes provide leukemia cells with critical substrates for the trichloroacetic acid cycle and the synthesis of nonessential amino acids, both of which reproduce α-KG to maintain its cellular level. In xenogeneic transplantation experiments, deprivation of BCAA from daily diet strongly inhibited expansion, engraftment and self-renewal of human acute leukemia cells. Inhibition of BCAA catabolism in primary AML or ALL cells specifically inactivates the function of the polycomb repressive complex 2, an epigenetic regulator for stem cell signatures, by inhibiting the transcription of PRC components, such as zeste homolog 2 and embryonic ectoderm development. Accordingly, BCAA catabolism plays an important role in the maintenance of stemness in primary human AML and ALL, and molecules related to the BCAA metabolism pathway should be critical targets for acute leukemia treatment.

Simultaneous Inhibition of Ceramide Hydrolysis and Glycosylation Synergizes to Corrupt Mitochondrial Respiration and Signal Caspase Driven Cell Death in Drug-Resistant Acute Myeloid Leukemia.

Acute myelogenous leukemia (AML), the most prevalent acute and aggressive leukemia diagnosed in adults, often recurs as a difficult-to-treat, chemotherapy-resistant disease. Because chemotherapy resistance is a major obstacle to successful treatment, novel therapeutic intervention is needed. Upregulated ceramide clearance via accelerated hydrolysis and glycosylation has been shown to be an element in chemotherapy-resistant AML, a problem considering the crucial role ceramide plays in eliciting apoptosis. Herein we employed agents that block ceramide clearance to determine if such a "reset" would be of therapeutic benefit. SACLAC was utilized to limit ceramide hydrolysis, and D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-threo-PDMP) was used to block the glycosylation route. The SACLAC D-threo-PDMP inhibitor combination was synergistically cytotoxic in drug-resistant, P-glycoprotein-expressing (P-gp) AML but not in wt, P-gp-poor cells. Interestingly, P-gp antagonists that can limit ceramide glycosylation via depression of glucosylceramide transit also synergized with SACLAC, suggesting a paradoxical role for P-gp in the implementation of cell death. Mechanistically, cell death was accompanied by a complete drop in ceramide glycosylation, concomitant, striking increases in all molecular species of ceramide, diminished sphingosine 1-phosphate levels, resounding declines in mitochondrial respiratory kinetics, altered Akt, pGSK-3β, and Mcl-1 expression, and caspase activation. Although ceramide was generated in wt cells upon inhibitor exposure, mitochondrial respiration was not corrupted, suggestive of mitochondrial vulnerability in the drug-resistant phenotype, a potential therapeutic avenue. The inhibitor regimen showed efficacy in an in vivo model and in primary AML cells from patients. These results support the implementation of SL enzyme targeting to limit ceramide clearance as a therapeutic strategy in chemotherapy-resistant AML, inclusive of a novel indication for the use of P-gp antagonists.

Dual mTORC1/2 Inhibition Synergistically Enhances AML Cell Death in Combination with the BCL2 Antagonist Venetoclax.

Acute myelogenous leukemia (AML) is an aggressive disease with a poor outcome. We investigated mechanisms by which the anti-AML activity of ABT-199 (venetoclax) could be potentiated by dual mTORC1/TORC2 inhibition. Venetoclax/INK128 synergism was assessed in various AML cell lines and primary patient AML samples in vitro. AML cells overexpressing MCL-1, constitutively active AKT, BAK, and/or BAX knockout, and acquired venetoclax resistance were investigated to define mechanisms underlying interactions. The antileukemic efficacy of this regimen was also examined in xenograft and patient-derived xenograft (PDX) models. Combination treatment with venetoclax and INK128 (but not the mTORC1 inhibitor rapamycin) dramatically enhanced cell death in AML cell lines. Synergism was associated with p-AKT and p-4EBP1 downregulation and dependent upon MCL-1 downregulation and BAK/BAX upregulation as MCL-1 overexpression and BAX/BAK knockout abrogated cell death. Constitutive AKT activation opposed synergism between venetoclax and PI3K or AKT inhibitors, but not INK128. Combination treatment also synergistically induced cell death in venetoclax-resistant AML cells. Similar events occurred in primary patient-derived leukemia samples but not normal CD34+ cells. Finally, venetoclax and INK128 co-treatment displayed increased antileukemia effects in in vivo xenograft and PDX models. The venetoclax/INK128 regimen exerts significant antileukemic activity in various preclinical models through mechanisms involving MCL-1 downregulation and BAK/BAX activation, and offers potential advantages over PI3K or AKT inhibitors in cells with constitutive AKT activation. This regimen is active against primary and venetoclax-resistant AML cells, and in in vivo AML models. Further investigation of this strategy appears warranted.

The RNA m6A Reader YTHDF1 Is Required for Acute Myeloid Leukemia Progression.

The m6A reader YTHDF1 is required for progression of acute myelogenous leukemia and can be targeted with the FDA-approved drug tegaserod to suppress leukemia growth.

Defining Primary Vs Secondary AML: Correlation between Pathology and NGS/FISH

Introduction: The treatment of acute myelogenous leukemia (AML) is often determined by factors that include an antecedent dysplasia. At presentation it is often unknown whether a patient has had dysplastic bone marrow features or not. We opted to look back at our leukemia patients to determine the frequency of antecedent dysplasia and to determine if next generation sequencing (NGS) or Florescent in situ hybridization (FISH) findings correlate with the pathologic review. Methods: We chart reviewed 43 charts from a period of 9/21/2021 to current from Orlando Health records. We focused on 29 charts that we could gather complete information from. We reviewed initial bone marrow biopsies and NGS/ FISH findings. We determined if a patient had a known history of myelodysplasia from previous records. If there was no known history of myelodysplastic syndrome (MDS) but dysplasia was seen on the bone marrow biopsy, we looked at NGS/FISH. While there can be overlap with AML, findings that suggest MDS are as follows: NGS: TET2, NRAS, U2AF1, RUNX1, TP53, SF3B1 FISH: 5q-, -5 (5p15, 5q31, 5q33), 7q-, -7 (Cen 7, 7q22, 7q31), Trisomy 8 (Cen 8), MLL (11q23), 20q- (20q12, 20qter) We then examined what treatments were used based on these findings. Results: Overall: 11/29 patients had dysplasia mentioned in their initial pathology report. 4/11 had a known preexistent dysplasia. Of the 7 who didn't have known dysplasia, 6/7 had an NGS or FISH suggesting MDS. Age: Of the patient who had dysplastic findings as well as NGS/FISH to support those findings, the patients had an average age of 57. The one patient without MDS/FISH findings has an age of 69. The average age of all patients with dysplastic findings on their bone marrow was 60. Of the patients who did not have any dysplastic findings on the bone marrow, the average age was 58. Treatment: Vyexos (liposomal daunorubicin and cytarabine), a therapy generally used for secondary leukemias was only used in two patients. Age and performance status was cited as a reason to use alternative therapy. Although one patient did receive 7+3 (idarubicin and cytarabine) instead of Vyxeos. One patient also had severe COVID pneumonia at diagnosis as the reason for not getting induction with Vyexos and instead getting venetoclax/azacitidine. Four patients were either treated with hypomethylating agent of either azacitidine or decitabine, again this was normally chosen due to performance status, age, or active COVID pneumonia as above. Conclusion: These results show that pathology findings of underlying dysplastic changes seem to correlate with NGS or FISH findings. Of the 7 patients with these bone marrow changes but no known MDS, 6 of them had NGS or FISH to support an underlying disease. Factors such as age did not help to predict antecedent MDS. Based on this information it may be appropriate to start with induction therapies such as Vyxeos when dysplastic changes are seen on the bone marrow while FISH and NGS are still pending, if their performance status and age allow to do so. Further larger retrospective studies to compare underlying dysplastic findings versus FISH/NGS findings should be done to further support this concept.

Novel Small Molecule and Peptide Inhibitors of CREB in Leukemia Cells

Background: Acute Lymphoblastic Leukemia (ALL) and Acute Myelogenous Leukemia (AML) account for nearly 45% of all childhood cancer. Current therapy for acute leukemia results in significant morbidity and late complications in children. Therefore, more effective and less toxic therapies to treat leukemia are needed. Previous work in our laboratory has identified CREB (cAMP Response Element Binding Protein), a transcription factor that promotes cell proliferation and survival in acute leukemia, is overexpressed in primary ALL and AML cells and is a potential target for therapy. We have investigated small molecules and peptides to inhibit the critical interaction between the phosphorylated KID (Kinase-Inducible) domain of CREB and the KIX (KID-interacting) domain of the coactivator CBP (CREB Binding Protein). Methods: We assessed a library of previously untested alpha-mimetic compounds from ChemDiv designed to mimic interactions between α-helices and protein surfaces for predicted interaction with a shallow hydrophobic groove on the interaction surface of KIX. We interrogated the 24,737 compounds in the library using rigid-receptor ligand docking, followed by induced-fit ligand docking of the top compounds using two different software, MOE and Schrodinger's Bioluminate. The top 50 binding poses from each software's rankings were combined to yield 87 candidate alpha-mimetic molecule inhibitors. In addition to our screen of small molecules, we have designed peptide inhibitors that closely mimic the αB helix of pKID. We investigated two methods to enhance the stability and cell-penetrating ability of our peptides: hydrocarbon stapling and the utilization of a rigid scaffold from the scorpion toxin peptide BmBKTtx1. Based on the calculated binding scores and locations, the most promising chimeric peptide designs were further investigated for binding stability using 500 nanosecond molecular dynamics (MD) simulations modeling their interaction with KIX. Results: The binding poses of the final set of small molecules were visually inspected for occupation of key hotspots on the pKID:KIX interaction surface, including the location of the phosphorylation sight of KID. Of the 87 molecules and their binding poses, only 21 of them (24%) obstructed fewer than two of the three hotspots chosen for the analysis. Moreover, 80 of the molecules (92%) extended into the hydrophobic pocket, the most critical hotspot. The multi-residue construction of this pocket likely contributed to the high similarity in interaction fingerprint between these molecules and pKID; occupation of this pocket can serve as a good indicator of strong ligand binding to KIX in future assays. The most-promising peptide designs using the scorpion toxin scaffold were assessed with MD simulations and compared to a control peptide comprised primarily of the αB helix of pKID. The chimeric peptides showed increased binding stability over the control peptide with an average RMSD of 6.8Å vs. 12.5Å, illustrating the benefits of the stability afforded by the scorpion scaffold design. Several of the hydrocarbon stapled peptides were tested in vitro using fluorescence polarization (FP) assay developed by our lab (Chae et al. Bioorganic & Medicinal Chemistry Letters, 2019) and in AML HL-60 and KG-1 cells and were shown to have growth inhibitory effects by Cell-Titer Glo Assays. Discussion: These small molecules and scorpion toxin-derived peptides represent novel candidate inhibitors of the pKID:KIX interaction implicated in leukemia. Based on promising results from our in silico analyses of these inhibitors, we have obtained ten structurally diverse alpha-mimetic small molecules and three chimeric peptides and aim to test their efficacy in vitro using the FP assay. Furthermore, we will obtain high resolution cryo-EM structures of successful compounds and peptides bound to the KIX domain using a double-shell protein scaffold (Zhang et al. ACS Central Science, 2022), and use these structures for further refinement of the inhibitors. The development of new inhibitors for the pKID:KIX interaction promises to reveal insights into the structural relationship between the two domains and the basis for effective, targeted leukemia therapeutics.

Targeting Vimentin (VIM) in Acute Myelogenous Leukemia (AML)

Background: Vimentin (VIM) is a type III intermediate filament (IF) that anchors organelles in the cell, maintains cell integrity, and facilitates mitosis. A 2018 study demonstrated that the up-regulation of VIM is associated with poor clinical outcome in patients with Acute Myeloid Leukemia (AML). This study also reported that VIM regulates signaling networks that promote leukemic cell survival while conferring resistance to various stressors. A separate study revealed that VIM over-expression was associated both with markedly shorter overall survival (OS) and leukemia-free survival (LFS) in adult AML patients. VIM has been shown to be targetable by the steroidal lactone Withaferin-A which directly binds VIM, hyper-phosphorylates VIM's Serine 38 residue, and leads to an anti-proliferative effect characterized by perturbation of intermediary filaments. We investigated VIM expression and prognostic impact in pediatric AML, and whether inhibiting VIM affected chemosensitivity in vitro. Methods: Reverse Phase Protein Array (RPPA) was performed to quantify relative expression of 233 total and 63 post-translational modified proteins in 500 pediatric AML patients from the COG AAML1031 trial. AML cell lines with low VIM expression (OCI-2) and high VIM expression (OCI-3) as well as fresh, primary AML samples were exposed to the VIM inhibitor Withaferin-A alone, or in combination with Doxorubicin and Cytarabine chemotherapy. Other known VIM inhibitors including FiVe1, Simvastatin, and Ganoderic Acid A were tested for efficacy in our context, but only Withaferin-A produced a promising anti-leukemia effect. Results: In pediatric AML, VIM was over-expressed relative to normal CD34+ cells in all subtypes: bulk, CD3/CD19 depleted, CD34+, CD34+/38+, CD34+/38-, CD34-, CD34-/38+, and CD34-/38-. Pearson correlation analysis revealed significant negative correlations between VIM and proteins known to decrease during metastasis such as PDGFR-beta, TP15.pS15, HIF1-alpha and VHL. Elevated VIM correlated positively with HNRNPK, WTAP, ASH2L, and KDM1A, and H3K4Me2; alterations in histone methylation that are associated with metastatic potency. VIM expression was highest in FAB subtypes M4 and M5, and in patients with t(9;11) and MLL-rearrangements. VIM levels were lower in cytogenetically favorable and higher in unfavorable risk groups. Cumulative survival probability to standard chemotherapy progressively decreased as VIM expression increased (Fig. 1). We tested whether VIM inhibition with Withaferin-A at 50 nM and 100 nM, would affect proliferation or cell survival, finding a significantly stronger anti-proliferative effect (p<0.02) in the high-VIM OCI-3 cell line compared to the low-VIM OCI-2 cell line at 72 and 96 hours after treatment (Fig. 2). Combined with chemotherapy, Withaferin-A lead to greater cell death in the high-VIM OCI-3 cell line as opposed to low-VIM OCI-2 at 72 and 96 hours. For instance, in OCI-3, 100 nM Withaferin-A combined separately with either 1 uM Cytarabine or 50 nM Doxorubicin to decrease proliferation more than either chemotherapeutic agent alone (p=0.007 and p=0.0013). In primary AML samples, proliferation was slightly more inhibited at 72 hours post-treatment when Withaferin-A was paired with individual chemotherapeutics. Conclusions: Proteomic analysis demonstrated that VIM was elevated in numerous leukemia cell subtypes relative to normal CD34+ cells in children and adults and that prognosis worsened as VIM levels increased in pediatric AML. AML cell lines highly expressing VIM such as OCI-3 displayed sensitivity to both Withaferin-A monotherapy and Withaferin-A in combination with Cytarabine and Doxorubicin. Finally, Withaferin-A significantly reduced viability and proliferation after 72 hours of treatment in primary AML samples at select doses in combination with chemotherapeutics. These results suggest that targeting VIM could improve the efficacy of chemotherapy in high VIM expressing subpopulations and potentially improve overall survival. Withaferin-A's efficacy both as a mono-therapeutic and in combination with standard chemotherapeutics used to treat AML patients offers an exciting new area of research that requires further investigation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clofarabine and Busulfan Myeloablative Conditioning in Allogeneic Hematopoietic Cell Transplantation for Patients With Active Myeloid Malignancies

Patients with refractory or relapsed and refractory myeloid malignancies have a poor prognosis. Allogeneic hematopoietic cell transplantation (HCT) with myeloablative conditioning (MAC) in patients with active, chemotherapy-refractory myeloid disease is historically associated with high rates of relapse and nonrelapse mortality (NRM). A MAC regimen combining clofarabine with busulfan (Clo/Bu4) has been reported to exhibit antileukemic activity with acceptable toxicity in patients age ≤70 years. Here we describe the clinical outcomes of a real-world population of patients with active myeloid malignancies undergoing allogeneic HCT with Clo/Bu4 MAC. In a single-center retrospective descriptive analysis, we identified patients who underwent HCT for myeloid malignancies not in remission using Clo/Bu4 MAC between 2012 and 2020. We report event-free survival (EFS) and overall survival (OS), cumulative incidences of relapse and NRM, and the incidence and severity of acute and chronic graft-versus-host disease (GVHD). We identified 69 patients with a median age of 60 years (range, 22 to 70 years). Most patients had relapsed/refractory or primary refractory acute myelogenous leukemia (AML; n=55) or refractory myelodysplastic syndrome (MDS; n=12); 1 patient had chronic myelogenous leukemia, and 1 patient had a blastic plasmacytoid dendritic cell neoplasm. Fifty patients (72.5%) had complete remission at day 100 post-transplantation. Two-year EFS and OS were 30% (95% confidence interval [CI], 20% to 44%) and 40% (95% CI, 29% to 54%), respectively. Patients with AML had a 2-year EFS and OS of 28% (95% CI, 18% to 44%) and 38% (95% CI, 27% to 54%), respectively; those with MDS had a 2-year EFS and OS of 47% (95% CI, 25% to 88%) and 56% (95% CI, 33% to 94%), respectively. The cumulative incidence of relapse at 2 years was 39% (95% CI, 27% to 51%) for all patients, including 45% (95% CI, 31% to 58%) in the patients with AML and 18% (95% CI, 2% to 45%) in those with MDS. NRM at 2 years was 31% (95% CI, 20% to 42%), including 27% (95% CI, 15% to 39%) in patients with AML and 35% (95% CI, 10% to 63%) in those with MDS. The total incidence of acute GVHD (aGVHD) of any severity was 80%, and the incidence of grade III-IV aGVHD was 22%. In patients who achieved remission, those who required systemic immunosuppression for aGVHD (58%) had poorer 2-year EFS (29% versus 54%; P=.05) and 2-year OS (39% versus 70%; P=.04) compared to those who did not. The 2-year cumulative incidence of chronic GVHD was 44% (95% CI, 28% to 58%). Clo/Bu4 MAC followed by allogeneic HCT for patients with active myeloid malignancies is an effective transplantation strategy for patients up to age 70, particularly those with advanced MDS. The high incidence of and poor outcomes associated with aGVHD highlight the importance of optimizing preventative strategies.

Expression of CD123 Related Long Non-coding RNA in Acute Myeloid Leukemia Bone Marrow Mononuclear Cells and Its Clinical Significance.

Acute myelogenous leukemia (AML) is a very common hematopoietic malignancy. Hematopoietic stem cell transplantation can improve the therapeutic effect of AML, but the 5-year survival rate is very low. CD123 imbalance, abnormal gene expression, and epigenetics play an important role in the pathogenesis of AML. This research was to explore the differential expression of CD123-related long non-coding RNA (lncRNA) in AML bone marrow mononuclear cells and provide a theoretical basis for targeted therapy of AML. High-throughput sequencing was performed to screen differentially expressed lncRNA in bone marrow mononuclear immunophenotypes of CD123+ and CD123- from patients with primary AML, and real-time quantitative PCR was adopted for screening and validation. There were 933 differentially expressed lncRNAs in the CD123+ group and the CD123- group, 407 lncRNAs were up-regulated and 463 lncRNAs were down-regulated in the CD123+ group. 14 lncRNAs with more than 2 times of difference were screened for identification, and it was found that compared with CD123- group, there was no substantial difference in the expression of JHDM1D-AS1, LINC01355, CASC15, FAM13A-AS1, HSPC324, LOC339803, LINC00877, and MAG12-AS3 in CD123+ group (P>0.05). The expressions of LOC101929698, BaALC-AS2, BOLA3-AS1, and FBX19-AS1 were considerably up-regulated (P<0.05), while the expressions of LOC100132249 and LINC02085 were considerably down-regulated (P<0.05). In summary, differentially expressed lncRNAs in bone marrow samples of CD123+ and CD123- group of newly diagnosed AML patients may be involved in the process of AML and seriously affect the prognosis of patients.

Characterizing Naked Nuclei Frequency and Movement in Primary AML Cell Culture Using an ECM-Based Model

INTRODUCTIONNaked nuclei (NN) are observed upon examining bone marrow aspirate slides from healthy individuals and from those with hematologic malignancies, but are not well understood. Without characteristic findings like cytoplasm or plasma membrane, NN are considered remnants of slide preparation or are discounted as cells of undetermined significance. NN have been associated with poor prognosis in several solid cancers. Understanding the significance of NN in hematologic malignancies such as acute myelogenous leukemia (AML) may elucidate valuable diagnostic and prognostic knowledge. Decellularized Wharton's jelly matrix (DWJM) is an extra cellular matrix (ECM)-based in vitro model that shares similar elements with the bone marrow ECM and can be used as scaffolding to culture leukemia cells. We hypothesize that NN exist in AML and interact with other cells, suggesting potential biological relevance within the bone marrow microenvironment.METHODSPrimary AML samples obtained by leukapheresis were cultured in suspension with growth media or in the presence of DWJM submerged in growth media. In samples grown with DWJM, cells that were non-adherent to the matrix were collected first and then adherent cells were isolated by treating DWJM with collagenase. Live cells were stained with CellVue Maroon (CVM) for membrane, CellTracker Green (CTG) for cytoplasm, and Hoechst 33342 for nucleus, followed by analysis with Amnis/Luminex ImageStream-X imaging flow cytometer (Figure 1A). NN were defined as events positive for nuclear stain and negative for cytoplasmic and membrane stains (Figure 1B). 3-D movement of adherent AML cells in DWJM was captured in real time using confocal microscopy. Fixed cells from leukemia cell line K562 served as a control for movement. NN (Hoechst positive only), non-nucleated (Hoechst negative/CTG positive), and nucleated cells (Hoechst and CTG positive) were identified by fluorescent labeling. NN were also observed after isolation by cell sorting. Cell speed, cell displacement from origin, and change in distance to closest neighboring cell over time were measured. Additionally, flow sorted NN were examined by immunohistochemistry (IHC).RESULTSAdherent populations contained significantly more NN than non-adherent and suspension populations. The frequency of NN in matrix adherent cells ranged from 0.4-2.4% at day 3 and 0.5-5.4% at day 7 of culture (Figure 1C). Through confocal microscopic analysis, we observed NN, nucleated, and non-nucleated cells moving at speeds ranging from 0.002-0.08 µm/sec. Fixed cells showed no discernible movement in DWJM. The average speed of NN [0.019 mm/s, SD 0.011] significantly differed from the average speed of nucleated and non-nucleated cells [0.027 mm/s, SD 0.016] (p=0.004) (Figure 1E). To demonstrate directional movement, we measured change in distance between NN and closest neighboring nucleated or non-nucleated cells over time. Cells (nucleated and non-nucleated) and NN moved closer to each other over time suggesting directional movement (p=0.001) (Figure 1D). NN also showed movement in DWJM after isolation by cell sorting. IHC analysis showed sorted NN stained positive for nuclear lamin A/C, which are considered markers of nuclear membrane (Figure 1F).CONCLUSIONSOur findings confirm that NN are present in primary AML cells cultured in vitro using ourECM-based model and that they can be isolated using flow cytometry. Additionally, NN display directional movement in DWJM suggesting that they interact with other cells and may be biologically relevant structures in the bone marrow microenvironment. [Display omitted] DisclosuresBaran: AstraZeneca/Acerta: Research Funding. Chu: Pfizer: Current equity holder in publicly-traded company; Acerta/AstraZeneca: Research Funding; TG Therapeutics: Research Funding.

Discovery and Pharmacological Characterization of JNJ-64619178, a Novel Small-Molecule Inhibitor of PRMT5 with Potent Antitumor Activity.

The protein arginine methyltransferase 5 (PRMT5) methylates a variety of proteins involved in splicing, multiple signal transduction pathways, epigenetic control of gene expression, and mechanisms leading to protein expression required for cellular proliferation. Dysregulation of PRMT5 is associated with clinical features of several cancers, including lymphomas, lung cancer, and breast cancer. Here, we describe the characterization of JNJ-64619178, a novel, selective, and potent PRMT5 inhibitor, currently in clinical trials for patients with advanced solid tumors, non-Hodgkin's lymphoma, and lower-risk myelodysplastic syndrome. JNJ-64619178 demonstrated a prolonged inhibition of PRMT5 and potent antiproliferative activity in subsets of cancer cell lines derived from various histologies, including lung, breast, pancreatic, and hematological malignancies. In primary acute myelogenous leukemia samples, the presence of splicing factor mutations correlated with a higher ex vivo sensitivity to JNJ-64619178. Furthermore, the potent and unique mechanism of inhibition of JNJ-64619178, combined with highly optimized pharmacological properties, led to efficient tumor growth inhibition and regression in several xenograft models in vivo, with once-daily or intermittent oral-dosing schedules. An increase in splicing burden was observed upon JNJ-64619178 treatment. Overall, these observations support the continued clinical evaluation of JNJ-64619178 in patients with aberrant PRMT5 activity-driven tumors.

Combinatorial Anti-Leukemic Activity Of Liposomal Ceramide and Inhibitors Of Autophagy

BackgroundCeramides are naturally occurring cellular sphingolipids which are increased following various effective cancer therapies. Bioavailable forms of ceramide are under development as therapeutic tools (Anticancer Agents Med Chem. 2011 Nov;11(9):911-9). Short side chain (C6) ceramide has, in pegylated nanoliposomal (NL) formulations, shown excellent cellular uptake and significant anti-tumor activity in a variety of tumor types and models. Cellular autophagy is an active microsomal process implicated in ceramide metabolism and potentially in the detoxification of C6-ceramide. We have previously shown synergistic combination therapy with Lip-C6-ceramide and vinblastine and its association with autophagy dysfunction in hepatocarcinoma and colorectal cancer models (Cancer Lett. 2013 Sep 1;337(2):254-65.). Methods and findingsWe explored the therapeutic activity of nano-liposomally delivered C6-ceramide (Lip-C6) in cell lines and primary human acute myelogenous leukemia (hAML). Lip-C6 cytotoxicity was tested in multiple human and murine AML lines (in short term viability assays) and in multiple cryopreserved primary human AML cases (in clonogenic assays - colony formation in methylcellulose). Lip-C6 demonstrated anti-AML activity in these cells with an EC50 of 2-23uM Lip-C6. In order to enhance this efficacy the interplay between ceramide induced apoptosis and autophagy was explored. The AML line KG1 was found to be relatively resistant to Lip-C6 (EC50 23uM). KGI showed supra-additive sensitivity to the combination of Lip-C6 with either vinblastine (VBL) or vincristine, both microtubule binding agents with the ability to reduce autophageic flux. KG1 sub-clones with knockdown of ATG5 demonstrated increased sensitivity to Lip-C6 relative to control or scrambled knockdown transfectants, consistent with the hypothesis that autophagy “rescues” this line from ceramide cytotoxicity. To further confirm these findings we examined the interaction of Lip-C6 (20uM) with VBL (5uM) or chloroquine (25uM) in a series of 13 fresh, patient derived AML cases (Figure: A-F). After 48 hours of co-culture, cells were examined for apoptosis of whole leukemic(E) and putative leukemia “stem cell” (LSC - F) fractions by flow cytometry. We also studied the in-vivo activity of Ghost (control) NLs, Lip-C6, VBL-NLs and combinatorial Lip-C6+VBL-NLs in NSG xenografts bearing a single poor prognosis (inversion 3) hAML (data are shown in the figure G.) [Display omitted] ConclusionsWe conclude that VBL and chloroquine both yield significant enhancement of Lip-C6 ceramide anti-hAML cytotoxicity. Available evidence suggests that this effect is mediated by inhibition of autophagy in these ceramide stressed cells. These data suggest that the combination of Lip-6 with autophagy inhibitors delivered simultaneously or in combinatorial formulations may prove clinically useful for these disorders. Disclosures:Kester:Keystone Nano Inc.: Membership on an entity's Board of Directors or advisory committees, Penn state research foundation has licensed ceramide nanoliposome technology to keystone nano Inc. For commercialization, MK is co-founder and CMO of Keystone Nano., Penn state research foundation has licensed ceramide nanoliposome technology to keystone nano Inc. For commercialization, MK is co-founder and CMO of Keystone Nano. Patents & Royalties.

Suppression of mTOR Signaling Attenuates Apoptotic Effects Induced By the NEDD8-Activating Enzyme Inhibitor Pevonedistat (MLN4924) in Acute Myeloid Leukemia Cells

Acute myelogenous leukemia (AML) is a heterogeneous disease and often relapses after standard chemotherapy. Therefore, novel therapies for AML are needed. Recently, the NEDD8 conjugation pathway and the mammalian target of rapamycin (mTOR) signaling pathway have emerged as important regulatory pathways for cancer therapy. Since some components of the mTORC1/2 pathways such as Deptor are neddylation targets, we postulated that combined exposure to NEDD8-activating enzyme (NAE) inhibitor pevonedistat and the dual mTORC1/2 inhibitor sapanisertib (TAK-228) might have additive or synergistic effects in AML.To test pevonedistat and sapanisertib effects on AML cells, we used the agents alone or in combination followed by assessment of proliferation, apoptosis and signal transduction in human AML cell lines as well as in primary cells. By AnnexinV/7AAD flow cytometry analysis, we found that pevonedistat significantly induced AML cell death and apoptosis in a time- and dose-dependent manner. Western blot showed that poly (ADP-ribose) polymerase (PARP) and caspases 3/8/9 were cleaved, indicating that pevonedistat-treated cells were actively undergoing apoptosis. Among the six AML cell lines (HL-60, KG1a, Molm-13, MV4-11, THP-1 and U937) tested, MV4-11 was the most sensitive cell line (IC50 <25nM) and KG1a was the least sensitive cell line (IC50 >1000nM) after 48 hours of exposure. The average IC50 on primary AML blast cells (5 primary samples) was 400nM at 48 hours. Western blot analysis showed that the neddylated cullin levels were markedly decreased confirming inhibition of the NEDD8 conjugation pathway by pevonedistat. We also found that pevonedistat induced morphologic changes and increased CD11b expression in Molm-13 and MV4-11 cells, indicating cell differentiation.The effects of pevonedistat on AML cells could be attenuated by pre- or co-treatment with the mTORC1/2 inhibitor sapanisertib. We found that sapanisertib alone induced limited cell death in most AML cell lines and primary AML patient blasts. The results from XTT assay and flow cytometry analysis indicated that sapanisertib alone significantly decreased cell size, arrested cells in G0-G1 phase and reduced protein content of AML cells but did not induce robust apoptosis. This suggested that through inactivating mTOR signal pathway, sapanisertib suppressed cell activity and induced a dormant/quiescent status and thereby attenuated the apoptotic effects of pevonedistat. With concurrent pevonedistat and sapanisertib exposure, the expression of apoptotic mediators such as cleaved PARP and caspases 3 were decreased compare to pevonedistat treatment alone (See Figure).To confirm the role of mTOR inhibition on pevonedistat-induced AML apoptosis, we targeted mTORC1/2 by knocking down mTOR expression with small hairpin RNAs (shRNA). Downregulation of mTOR expression mirrored the effects of sapanisertib. mTOR inhibition by shRNAs resulted in decreased AML cell size and arrest of cells in G0-G1 phase. In addition, pevonedistat-induced cell death was significantly impeded in the cells infected with mTOR shRNA lentivirus compared to scrambled shRNA (p<0.01). Therefore, we validated that mTOR signal inactivation protects AML cells from NAE inhibitor pevonedistat-induced apoptosis using both pharmaceutical and genetic approaches. To further study the mechanism behind mTOR inhibition inducing resistance to pevonedistat, we tested rapamycin, an mTORC1 inhibitor. Concurrent exposure to rapamycin did not significantly alter the effects of pevonedistat and did not change cell size and cell cycle status. In addition, rapamycin did not inhibit the phosphorylation of 4E-BP1, suggesting that mTORC1 inhibition alone may be insufficient to inhibit the effects of pevonedistat, and suggesting a role for mTORC2.Taken together, our data demonstrate that the NEDDylation pathway inhibitor pevonedistat has strong potential as a therapeutic agent for AML therapy via apoptosis induction. However, when AML cells are arrested in G0-G1 with reduced proliferation rates by dual mTORC1/2-inhibition, the ability of pevonedistat to induce apoptosis is suppressed. Whether this is due solely to the cytostatic effects of dual mTOR inhibitors or also involves other mechanisms remains uncertain. While both sapanisertib and pevonedistat have clear anti-leukemia effects, their concurrent use may inhibit NAE inhibitor-induced apoptosis. [Display omitted] DisclosuresLiesveld:Onconova: Honoraria; Seattle Genetics: Honoraria.

IQGAP1: A Potential Therapeutic Target in Acute Myelogenous Leukemia (AML)

Introduction: While AML is phenotypically diverse, genome-wide sequencing studies indicate that the median number of non-synonymous mutations is eight (Science 339,1546, 2013) and that the same pathways are affected in tumors with distinct genetic alterations (Nature 453,1112-1116, 2008). These insights provided the impetus to immunoaffinity purify shared antigenic moieties in human AML and examine their prevalence as well as function.Methods: We employed a previously described method wherein immunization of rabbits with normal human peripheral blood cells whose surface charge had been modified by incubation with fluorodinitrobenzene (FDNB) elicited an antibody response that cross reacted against a broad range of human leukemias (Nature 232,197, 1971). Replicating the described methods WBCs from healthy volunteer donors were suspended at 1x107 cells per ml of PBS and mixed with equal volume of 0.86 mM FDNB for 12 to 15 minutes at room temperature. Three rabbits were immunized with FDNB-treated cells (experimental rabbits). A control rabbit was immunized with sham-treated cells (control rabbit). After complement inactivation, immune sera were absorbed against WBCs from healthy donors. Absorbed immune sera were tested in clinical AML samples by Western blotting. Immunoprecipitation of antigens from whole cell lysates of clinical AML samples was performed using IgG adsorbed on protein A/G Agarose beads. Liquid chromatography and mass spectrometry of the immunoprecipitated material was performed. Fold change in IQGAP1 expression in normal vs AML bone marrow was determined by qRT-PCR and from raw data from Gene Expression Omnibus at the NCBI. IQGAP1 expression was evaluated by immunohistochemistry in cell lines, bone marrow biopsies from AML patients and normal healthy volunteers. IQGAP1 expression was modulated by RNAi and the effect on proliferation and colony formation was measured in cell lines.Results: Western blotting of whole cell lysates of clinical AML samples revealed bands that were recognized by immune serum from experimental rabbits but not the control rabbit. Immuno-precipitation of antigens from whole cell lysates of clinical AML samples revealed the scaffolding protein IQGAP1 as being differentially recognized in independent experiments. We focused on IQGAP1 because it regulates multiple cellular functions including signaling pathways involved in proliferation and transformation and stimulates cell motility and invasion. It is overexpressed in colon cancer, pancreatic cancer, hepatocellular carcinoma, glioma, and other cancers. (Cell Signaling 21,1471-1678, 2009). Western blots of human AML samples probed with anti-IQGAP1 antibody revealed the predicted 190 kDa band, see figure. There was 3 to 9-fold greater expression of IQGAP1 expression in AML versus normal bone marrow observed by qRT-PCR. Analysis of raw data from Gene Expression Omnibus at the NCBI revealed fold change of -3.22636, p-value 2.62 x 10e-7 between normal bone marrow versus AML. Preliminary IHC studies showed strong membranous staining in three leukemia cell lines and bone marrow biopsies from primary AML bone marrow samples but weak partial staining in normal bone marrow. Knocking down expression of IQGAP1 in K562, MV4-11 and THP1 cell lines resulted in significant decrease in proliferation and colony formation as compared to effects of scramble control. Mean and SD of colonies per 2000 cells plated were for K562: naïve 311+/-46, IQGAP1 knockdown 37+/-12, scramble control 127+/-12, MV4-11: naïve 156+/37, IQGAP1 knockdown 55+/- 8, scramble control 147+/-16, and THP1 naïve 232+/-45, IQGAP1 knockdown 17+/-2.9, scramble control 150+/-24.Conclusions: 1. The scaffolding protein IQGAP1is overexpressed in AML compared to normal bone marrow. 2. Knocking down expression of IQGAP1 in AML cell lines resulted in significant decrease in proliferation and colony formation providing proof of concept that IQGAP1 is a potential therapeutic target in AML. [Display omitted] DisclosuresLiesveld:Onconova: Honoraria; Seattle Genetics: Honoraria.

The Pharmacological Blockade of Phosphodiesterase IV Potentiates the Anti-Tumor Effects of Arsenic Trioxide (ATO) in Acute Myelogenous Leukemia Cells through Multiple Signaling Pathways

Preclinical in vitro and in vivo studies showed that Arsenic Trioxide (ATO) has antileukemic effects both as a single agent and in combination with conventional therapies or with other molecularly targeted agents . Furthermore, analogs of cAMP have been shown to enhance the therapeutic effects of ATRA and ATO in APL cells. Because cAMP pathway is modulated by various Inhibitiors of phosphodiesterase IV (PDEIV), some of which are already in the clinics for the treatment of asthma, the aim of this study was to investigate whether the pharmacological inhibition of PDEIV potentiates the cytodifferentiating and/or proapoptotic activities of ATO in acute myelogenous leukemia (AML) cells.We found that PDEIV inhibitors induced the upregulation of the myeloid markers (CD38 and CD11b), and potentiated the cytodifferentiating action of ATO in APL cells via induction of cAMP; the cytodifferentiating effects of the PDEIV inhibitors /ATO combination in leukemic cells was also confirmed by nitro blue tetrazolium reductase activity assay. Accordingly, we also found that the inhibition of PDEIV accelerated the ATO-induced degradation of PML-retinoic acid receptor α (PML-RARα) oncoprotein in NB4 cells, thus removing the PML-RARα-induced maturation blocks.Furthermore we also demonstrated that PDEIV inhibitors synergized with ATO to induce apoptosis in both APL NB4 and non-APL HL-60, MOLM-13 and OCI-AML-3 AML cell lines and significantly (P<.01) potentiated the ATO-induced cell death of fresh purified leukemic blasts in 8 out of 10 AML patients analyzed with different genetic abnormalities; in contrast, no significant cytotoxicity in peripheral blood mononuclear cells from 3 healthy volunteers was observed after PDEIV inhibitors/ATO treatment. We consistently found that the inhibition of PDEIV strongly potentiated the ATO-induced activation of caspase-3 and PARP fragmentation in all the tested cell lines as well as in primary AML blasts from patients, and, by using a peptide inhibitor approach, we demonstrated that caspase activity was indispensable for PDEIV inhibitors / ATO-induced apoptosis. Furthermore, siRNA knockdown of endogenous PDE4A and PDE4B gene expression recapitulated the ability of PDEIV inhibitors to sensitize leukemic cells to ATO, thereby confirming the significant role of these enzymes in mediating ATO sensitization of leukemic cells to PDEIV inhibitors /ATO-induced apoptosis. Interestingly, by using cAMP analogs and cAMP competitors we demonstrated that PDEIV inhibitors /ATO -induced apoptosis was cAMP independent.In order to investigate the molecular effectors involved in PDEIV inhibitors /ATO-induced apoptosis we first evaluated the effects of the combined treatment on Bcl-2 and p53 family proteins and their biologic relevance in the synergism observed between PDEIV inhibitors and ATO. By western blot analysis we demonstrated that co-treatment with PDEIV inhibitors and ATO increased protein expression of pro-apoptotic of Bax, Bak and/or Puma and decrease anti-apoptotic Mcl-1, A1/Bfl1 and/or Bcl-2; in agreement with the biochemical findings indicating the activation of the mitochondrial apoptotic pathway, we found that combined treatment with PDEIV inhibitors and ATO strongly potentiated mitochondrial depolarization induced by ATO alone in all the tested cell lines. Preliminary data indicate the involvement of p73 pathway which is consistent with the observed upregulation of Bax, Bak and/or Puma in AML cells; ongoing experiment are in progress to evaluate whether p53 pathway is activated by the combined treatment in MOLM-13 and OCI-AML-3 AML cell lines as well as in fresh purified leukemic blasts.Additionally, we found that co-treatment with PDEIV inhibitors and ATO also induced prosurvival signals by increasing ERK and Bad phosphorylation and the expression of anti-apoptotic Bcl-xL, and the pharmacological or genetic disruption of ERK or Bcl-xL consistently and significantly potentiated the cytotoxic effects of the combination PDEIV inhibitors /ATO. Altogether these findings suggest to extend the study to more patients and strongly support the rationale for testing these promising combinations in clinically relevant in vivo mouse models of drug-resistant leukemia. DisclosuresNo relevant conflicts of interest to declare.

A Unique N-Phenylbenzamide Inhibitor Targeting Non-Receptor Tyrosine Kinases Potently Suppresses AML Cell Growth In Vitro and In Vivo

Acute myelogenous leukemia (AML) is a hematologic cancer correlated with advancing age and poor prognosis. While diverse genetic changes are associated with AML, activating mutations in the Flt3 receptor tyrosine kinase are associated with nearly one-third of cases and include internal tandem duplication (ITD) of the regulatory juxtamembrane region. Additionally, recent studies have implicated several non-receptor tyrosine kinases in AML oncogenic signaling, including Fes, Syk and the myeloid Src-family members Fgr, Hck and Lyn. While selective Flt3 kinase inhibitors have been developed for clinical use, they have not produced durable responses, highlighting the need for new therapeutic approaches. In this study, we investigated the therapeutic potential of a multi-targeted tyrosine kinase inhibitor, TL02-59, which is based on an N-phenylbenzamide scaffold. Kinome-wide target specificity profiling (KINOMEscan) demonstrated a relatively narrow overall specificity profile for TL02-59, with only 23 potential targets out of 456 kinases when tested at a concentration of 1 µM. To confirm the results of the kinome-wide profiling against AML-associated kinases, we tested TL02-59 using in vitro kinase assays with recombinant Fes, Syk, Fgr, Hck, and Lyn as well as wild-type and ITD forms of the Flt3 kinase domain. TL02-59 displayed remarkable potency against the myeloid Src-family kinases Fgr and Lyn, with IC50values in the 100 pM range. TL02-59 also inhibited Fes, Syk, Hck and both forms of Flt3 in vitro, albeit with IC50 values in the 150-400 nM range. We then tested TL02-59 for growth-suppressive activity against Flt3-ITD+ AML cell lines, MV4-11 and MOLM-14, and the Flt3 wild-type cell line THP-1. Remarkably, TL02-59 inhibited the proliferation of MV4-11 cells with an IC50 value of 780 pM, with slightly lower potency against the MOLM-14 cells (IC50 = 3.5 nM). Quantitative real-time RT-PCR (qPCR) and immunoblot analysis confirmed that the AML-associated TL02-59 target kinases identified in vitro, including Fgr and Lyn, are expressed and active in MV4-11 and MOLM-14 cells. In contrast, TL02-59 had no effect on THP-1 cell growth up to 1 µM, consistent with transformation of these cells by kinase-independent mechanisms. We next examined the growth suppressive activity of TL02-59 in 26 bone marrow samples from AML patients, half of which expressed Flt3-ITD and half of which expressed wild-type Flt3. Twenty of the primary AML samples responded to TL02-59 treatment with growth inhibitory IC50 values in the nM range. We then determined the relative expression levels of all possible TL02-59 target kinases from the KINOMEscan analysis in these primary AML samples by qPCR. We discovered a striking correlation between TL02-59 sensitivity and expression levels of Fgr, Hck, and Lyn, supporting the idea that these Src-family members are the primary targets for TL02-59 in AML. In contrast, no correlation was observed with Flt3 expression, consistent with the observation that the four most sensitive patient samples were wild-type for Flt3. To test the efficacy of TL02-59 in vivo , we used immunocompromised (NSG) mice engrafted with MV4-11 AML cells as a model system. Two weeks after MV4-11 cell engraftment, mice were treated once daily with TL02-59 at 1 or 10 mg/kg by oral gavage for a total of 21 days. The mice were then sacrificed and evaluated for MV4-11 cells in their peripheral blood, spleen, and bone marrow by flow cytometry and IHC. At 1 mg/kg, TL02-59 reduced MV4-11 engraftment in the bone marrow by 20% and spleen engraftment by half, while suppressing the number of leukemia cells in the peripheral blood by 70% compared to vehicle control animals. At 10 mg/kg, TL02-59 reduced bone marrow AML cells by 70% and completely eliminated leukemic cells from the spleen and peripheral blood. Overall, these results demonstrate that TL02-59 is an effective inhibitor of AML cell growth in vitro and in vivo with a mechanism of action independent of Flt3 mutational status. The primary kinase targets for this inhibitor appear to be myeloid Src-family kinases, including Fgr, Lyn and Hck. DisclosuresNo relevant conflicts of interest to declare.

Platelet Microparticles Protect Acute Myelogenous Leukemia Cells against Daunorubicin-Induced Apoptosis.

Simple SummaryActivated or apoptotic platelets both shed platelet microparticles that are proven to be internalized by many different cell types, including cancer cells. Here, we have investigated whether platelet microparticles can transfer their contents to the monocytic leukemia cell line THP-1 and if this could change cell activity and resistance to chemotherapy. We show that platelet microparticles were internalized by THP-1 cells and that platelet-associated microRNAs were elevated after a brief co-incubation. Furthermore, differentiation toward macrophages was induced and cell cycle progression, proliferation, and mitochondrial activity were decreased. Co-incubation with platelet microparticles increased chemotherapy resistance, which also was evident in acute myelogenous leukemia cells from patient samples, and it could be explained by the decrease in cell activity. Thus, platelet microparticles may have a role in the evolution of acute myelogenous leukemia and contribute to development of chemotherapy resistance, making them an interesting target for treatment.The role of platelets in cancer development and progression is increasingly evident, and several platelet–cancer interactions have been discovered, including the uptake of platelet microparticles (PMPs) by cancer cells. PMPs inherit a myriad of proteins and small RNAs from the parental platelets, which in turn can be transferred to cancer cells following internalization. However, the exact effect this may have in acute myelogenous leukemia (AML) is unknown. In this study, we sought to investigate whether PMPs could transfer their contents to the THP-1 cell line and if this could change the biological behavior of the recipient cells. Using acridine orange stained PMPs, we demonstrated that PMPs were internalized by THP-1 cells, which resulted in increased levels of miR-125a, miR-125b, and miR-199. In addition, co-incubation with PMPs protected THP-1 and primary AML cells against daunorubicin-induced cell death. We also showed that PMPs impaired cell growth, partially inhibited cell cycle progression, decreased mitochondrial membrane potential, and induced differentiation toward macrophages in THP-1 cells. Our results suggest that this altering of cell phenotype, in combination with decrease in cell activity may offer resistance to daunorubicin-induced apoptosis, as serum starvation also yielded a lower frequency of dead and apoptotic cells when treated with daunorubicin.

Combination of azacitidine and enasidenib enhances leukemic cell differentiation and cooperatively hypomethylates DNA

Azacitidine and enasidenib are two therapies available for treatment of acute myelogenous leukemia (AML), and the mechanisms of action of these drugs involve alteration of aberrant DNA methylation. We hypothesized that a combination of these agents could have interactive effects on DNA methylation and enhance differentiation in mIDH2 cells. Combination treatment enhanced cellular differentiation in TF-1 cells overexpressing IDJ2R140Q through increased hemoglobinization and increased hemoglobin γ RNA expression compared with the effects of single agents. Furthermore, in primary AML samples (IDH2R140Q or R172K), combination treatment reduced CD34+ cells and increased CD15+ cells to a greater extent than attained with single agents. To explore the mechanism of enhanced differentiation with combination treatment, the TF-1 epigenome was analyzed by profiling 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) DNA methylation changes. Enasidenib treatment alone increased 5hmC, consistent with reactivation of ten-eleven-translocation (TET) enzyme activity. Compared with treatment with azacitidine alone, combination treatment reduced 5mC levels at greater numbers of sites and these loci were significantly enriched in regions with increased 5hMC (25.8% vs. 7.4%). Results are consistent with a model in which enasidenib-mediated reactivation of ten-eleven-translocation enzymes cooperates with azacitidine-mediated inhibition of DNA methyltransferase enzymes, leading to greater reductions in DNA methylation and enhanced erythroid differentiation.

Immunoglobulin Superfamily Member 8 Is Indispensable for Myeloid Leukemia Via Wnt/β-Catenin Signaling Pathway

Immunoglobulin superfamily member 8 (IGSF8, also known as EWI-2, PGRL, and CD316), is a cell surface protein containing 4 immunoglobulin domains. IGSF8 directly binds to the tetraspanin molecules, CD9 and CD81, and modulates cell adhesion, migration, and growth. Previous studies demonstrated that IGSF8 was associated with prognosis and metastasis in several solid tumors. However, the role of IGSF8 in normal hematopoiesis and myeloid leukemia is still unclear. First, we examined the expression levels of Igsf8 in various hematopoietic fraction of wild-type murine bone marrow cells, and found that Igsf8 is expressed in all hematopoietic lineages. To investigate hematopoietic functions of Igsf8, we generated hematopoietic cells specific Igsf8 deleted mice (Igsf8fl/fl; Vav-Cre) and tamoxifen induced Igsf8 deleted mice (Igsf8fl/fl; Rosa26-CreERT). Igsf8fl/fl, Vav-Cre (denoted as Igsf8-/-) mice represented normal maturation. Deletion of Igsf8 did not significantly affect adult hematopoiesis in peripheral blood and bone marrow. Igsf8-/- long-term hematopoietic stem cells (LT-HSCs: CD34- Flk2- c-Kit+ Sca-1+ Lineage- cells) reduced colony forming ability in vitro, and serial competitive transplantation assay showed comparable donor chimerism by 3 months, but led to decrease Igsf8-/- donor chimerism at 4 months and those after second transplantation in vivo. These results suggest that Igsf8 does not affect the adult hematopoiesis, but it can affect their proliferative and reconstitutive capacity of HSCs. To investigate the effects of Igsf8 on myeloid leukemia, we generated MLL-AF9 and NRASG12V-driven acute myelogenous leukemia (AML), or BCR-ABL and NUP98-HOXA9-driven blast crisis of chronic myelogenous leukemia (CML-BC) mice models. Igsf8-/- led to a dramatic reduction in the number of leukemic colonies formed in vitro (Figure 1A). Igsf8-/- leukemia mice showed significantly longer survival in vivo (Figure 1B). This effect was also observed by eliminating Igsf8 expression after leukemia establishment using conditionally deletion. Igsf8-/- AML cells showed decreased S phase fraction. Igsf8-/- leukemia stem cells (LSCs: c-Kit+ Lineage- cells) triggered an increment of the apoptosis, which contribute to significantly lower proportion of LSCs in spleen of Igsf8-/- leukemic mice. Given that Igsf8-/- did not affect homing ability of leukemia cells, these results indicate that Igsf8 is required for propagation of myeloid leukemia and maintenance of LSC. To understand the Igsf8-mediated regulation of myeloid leukemia, we conducted RNA sequencing analysis of LT-HSCs, and LSCs of AML and CML-BC. Gene set enrichment analysis exhibited increase apoptosis related genes and decrease Wnt/β-catenin related genes in Igsf8-/- leukemic cells, but not in LT-HSCs (Figure 1C). Increment of pro-apoptosis genes, and decrement of anti-apoptosis genes and Wnt/β-catenin target genes in Igsf8-/- AML stem cells were validated in quantitative polymerase chain reaction analysis. Further, expression levels of β-catenin protein in Igsf8-/- leukemic cells were significantly lower compared to Igsf8+/+ leukemic cells, but not in normal hematopoietic stem and progenitor cells (Figure 1D). These results suggest that Igsf8 might be critical for myeloid leukemia maintenance via Wnt/β-catenin signaling pathway. Then, we investigated the effects of IGSF8 on human myeloid leukemia. We confirmed IGSF8 expression in several human myeloid leukemia cell line and primary patient-derived leukemia cells. Knockdown of IGSF8 by small hairpin RNA in myeloid leukemia cell lines (THP-1, MV4-11, SKM-1, and K562) and primary patient-derived AML cells exhibited reduced numbers of colony forming cells in vitro. Knockdown of IGSF8 also caused decrease expression of β-CATENIN in AML cell lines. These results indicate that IGSF8 is also required for propagation of human myeloid leukemia cells. Taken together, our present study reveals that Igsf8 is indispensable for myeloid leukemia, but not adult hematopoiesis, suggesting that IGSF8 inhibition should be considered for targeting myeloid leukemia. Disclosures Jimbo: Japan Society for the Promotion of Science: Research Funding. Konuma:SGH Foundation: Research Funding; The Japanese Society of Hematology: Research Funding; Institute for Frontier Life and Medical Sciences, Kyoto University: Research Funding. Ito:Institute for Frontier Life and Medical Sciences, Kyoto University: Research Funding.