Primary Acute Myelogenous Leukemia Samples Research Articles

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.

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

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.

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.

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.

Transcription factor Oct1 protects against hematopoietic stress and promotes acute myeloid leukemia

A better understanding of the development and progression of acute myelogenous leukemia (AML) is necessary to improve patient outcome. Here we define roles for the transcription factor Oct1/Pou2f1 in AML and normal hematopoiesis. Inappropriate reactivation of the CDX2 gene is widely observed in leukemia patients and in leukemia mouse models. We show that Oct1 associates with the CDX2 promoter in both normal and AML primary patient samples, but recruits the histone demethylase Jmjd1a/Kdm3a to remove the repressive H3K9me2 mark only in malignant specimens. The CpG DNA immediately adjacent to the Oct1 binding site within the CDX2 promoter exhibits variable DNA methylation in healthy control blood and bone marrow samples, but complete demethylation in AML samples. In MLL-AF9-driven mouse models, partial loss of Oct1 protects from myeloid leukemia. Complete Oct1 loss completely suppresses leukemia but results in lethality from bone marrow failure. Loss of Oct1 in normal hematopoietic transplants results in superficially normal long-term reconstitution; however, animals become acutely sensitive to 5-fluorouracil, indicating that Oct1 is dispensable for normal hematopoiesis but protects blood progenitor cells against external chemotoxic stress. These findings elucidate a novel and important role for Oct1 in AML.

Targeting the Second Bromodomain (BDII) of BET Family with Abbv-744 Results in Robust Anti-Leukemia Activity in AML Models with Excellent In Vivo Tolerability

The BET family proteins consist of BRD2, BRD3, BRD4 and BRDT, with each of these proteins containing two highly conserved bromodomains (BDI and BDII). First generation BET inhibitors, including ABBV-075, are pan-BET inhibitors that bind with nearly equimolar affinity to BDI and BDII. Pan-BET inhibitors generally block transcription and inhibit the proliferation of a large spectrum of tumor types, but exhibit a narrow therapeutic index. ABBV-744 is a highly selective inhibitor for the BDII of BET family proteins, exhibiting greater than 300-fold more potent binding affinity to the BDII bromodomain of BRD4 relative to BDI (Warren Kati AACR 2018; Xiaoyu Lin AACR 2018). In this study, we evaluated the anti-leukemia activities of ABBV-744 in acute myelogenous leukemia (AML) cell lines and primary samples.In AML cell lines, ABBV-744 induced G1 cell cycle arrest and apoptosis with caspase-3 activation and PAPR cleavage through regulation of the key proteins that are involved in cell cycle and apoptosis pathways (e.g. (BCL-2, BCL-XL, MCL-1, c-Myc). Mechanistically, ABBV-744 displaced BRD4 from the regulatory region of BCL2, RUNX1, BCL2L1 and IL8 genes by ChiP-Seq. MLL-rearranged cells were most sensitive to BDII inhibition, and ABBV-744 displaced both, BRD4 and MLL from the regulatory regions of BCL2 and c-Myc genes in MV4;11 (MLL-AF4) and Nomo-1 (MLL-AF9) cells. We examined anti-leukemia activity of ABBV-744 in 12 primary AML samples with diverse genetic alterations. ABBV-744 inhibited cell growth in 8/12 primary samples (20.2%-77.9%) and induced apoptosis (26.7 ± 9.6% in ABBV-744 group vs 14.1± 3.9% in DMSO group, p=0.007, Fig 1A). We next performed the whole genome transcriptome analysis of pre-treatment AML cells by RNA-sequencing (RNA-seq). The samples which were sensitive to ABBV-744 tend to have higher pro-apoptosis (TXNIP, DR5) and lower anti-apoptosis (BCL2, EXPO) than resistant samples.For efficacy assessment in vivo, we established a patient-derived xenograft (PDX) from an AML patient with FLT3-ITD, DNMT3A, IDH1 and NPM1 mutations in NSG mice. Upon engraftment, mice were randomized to receive vehicle or single agent ABBV-744 at a well-below MTD dose of 9.4 mg/kg for 21 days. The median survival time of mice treated with ABBV-744 (Median survival: 76 days) was significantly higher compared to untreated mice (Median survival: 67.5 days, p=0.007, Fig 1B). Next, the anti-leukemia efficacy of ABBV-744 was tested in 7 additional AML PDX models. NSG mice were injected with 7 AML PDX (one mouse per cohort design). After engraftment was confirmed by retro-orbital bleeding, mice were treated with vehicle or ABBV-744. Circulating tumor burden was measured by flow cytometry in peripheral blood samples from mice collected on indicated days of therapy. In five of the 7 models, ABBV-744 treatment delayed AML progression compared to untreated mice (survival days: 135 vs 105, 205 vs 153, 62 vs 46, 118 vs 77, 97 vs 86). No significant impact on mice' weight was noted (Fig 1C), and no clinical signs of toxicity recorded over the course of therapy.In summary, blockade of BET family bromodomain BDII promotes apoptotic cell death and suppresses proliferation in the majority of primary AML cells. Importantly, ABBV-744 therapy reduced AML tumor burden and extended survival in AML PDX models at a well-below MTD dose. Together, these results provide proof of concept that highly selective inhibitors of the second bromodomain of BET family may maintain robust anti-leukemia efficacy in AML while exhibiting improved tolerability relative to pan-BET inhibitors. [Display omitted] DisclosuresUziel:AbbVie Inc.: Employment. Shen:AbbVie Inc: Employment. Konopleva:Stemline Therapeutics: Research Funding. Lin:AbbVie Inc: Employment. Lu:AbbVie Inc: Employment. Zhang:AbbVie Inc: Employment.

Preclinical activity of FF-10501-01, a novel inosine-5′-monophosphate dehydrogenase inhibitor, in acute myeloid leukemia

BackgroundFF-10501-01 is a selective inosine monophosphate dehydrogenase (IMPDH) inhibitor that has shown activity in cancer cell lines. We studied whether FF-10501-01 is effective in targeting a variety of hypomethylating agent (HMA)-sensitive and −resistant acute myelogenous leukemia (AML) cell lines. MethodsWe treated multiple cell lines (including HMA-resistant cells) with FF-10501-01 and analyzed proliferation, apoptosis, and cell cycle status. We also assessed HMA-FF-10501-01 combinations and the ability of extracellular guanosine to rescue cell proliferation in FF-10501-01-treated cells. We performed high-performance liquid chromatography (HPLC) to study guanine nucleotide levels in treated and untreated cells. Finally, we studied the effects of FF-10501-01 in fresh peripheral blood cells taken from AML patients. ResultsFF-10501-01 showed a strong dose-dependent effect on proliferation and induced apoptosis at approximately 30μM. The effects of FF-10501-01 treatment on cell cycle status were variable, with no statistically significant trends. Guanosine rescued proliferation in FF-10501-01-treated cells, and HPLC results showed significant decreases in phosphorylated guanosine levels in MOLM13 cells. FF-10501-01 effectively reduced proliferation at concentrations of 300μM and above in 3 primary AML samples. ConclusionsFF-10501-01 effectively induces AML cell death and reduces AML peripheral blood cell proliferation by targeting guanine nucleotide biosynthesis regardless of HMA resistance status.

Abstract 3834: scFv antibodies against EphA2 receptor as a therapeutic strategy for AML

Abstract The ephrin (Eph) receptors have been sought as therapeutic targets in liquid and solid tumors. Eph receptors are a subcategory of receptor tyrosine kinases and play vital parts in cell survival and function. Recently, monoclonal antibodies have been designed to bind to the ligand-binding cavity, that block ephrin to bind to ephrin type-A receptor 2 (EphA2). The potential clinical importance has been demonstrated in lymphomas, and the ability of the antibody to reduce proliferation and induce apoptosis (Goldgur et. al 2014). Acute myelogenous leukemia (AML) is a disease with dismal outcomes and new therapeutic targets have been explored. EphA2 has been described to be critical for the survival in EphA2-positive mouse leukemia models and not essential for normal hematopoiesis. Thus, we examined cell surface expression of EphA2 in peripheral blood, and bone marrow samples from AML patients. We screened 30 primary AML samples and 5 normal samples and assessed their expression of EphA2 using flow cytometry using the lymphoma cell line (Raji) as a positive control, as reported to express EphA2. The EphA2 expression was evaluated under culture conditions as well as without culture. Prior to culture, 22 (73.3%) of 30 samples were EphA2 positive in more than 90% of blasts. The mean cell surface expression of EphA2 in blasts was 95.4% (range: 64.4-99.7%) with a mean fluorescence intensity (MFI) of 201.5 (range: 18.4-675.0) with a MFI of 4.4 for isotype control. Interestingly, we observed 13.3% increase in EphA2 expression post-culture, bringing the proportion of EphA2-high samples from 73.3% to 86.6% of total samples. In addition to the increase of EphA2-high population, the average MFI of this cohort was also increased by 47.6% post-culture relative to pre-culture. Furthermore, we saw a more significant increase in the CD34+ population and little or no change in the CD34- population. These data shows that many AML express EphA2, and that culturing with cytokines can induce robust EphA2 expression. After validating EphA2 expression in leukemia cells, we have tested a set of single-chain antibodies (scFv) against EphA2 that have shown activity against Raji cells (Goldgur et. al 2014). We tested two single-chain variable fragments (G2 or D2) on samples with low or high expression of EphA2. The samples were incubated in the serum free IMDM medium supplemented with cytokines. The scFv antibodies were tested at 0.5ug and 1.0ug and evaluated after 24hr- or 72hr-culture. We found that exposure to D2 resulted in a significant decrease in cell number after 72 hr-culture, 52.6% and 99.3% relative to vehicle control treatment for 0.5ug and 1.0ug respectively in EphA2-high AML cells. These data suggests that the scFv antibodies against EphA2 are lethal to AML cells that express high level of EphA2. Taken together, our data suggests that more than 70% of AML cells highly express EphA2 and can be targeted using scFv antibodies specific to EphA2. Citation Format: Michael Albert, Mayumi Sugita, Hongliang Zong, Juha Himanen, Monica L. Guzman. scFv antibodies against EphA2 receptor as a therapeutic strategy for AML. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3834.

Phase Ib/2 study of venetoclax with low-dose cytarabine in treatment-naive patients age ≥ 65 with acute myelogenous leukemia.

7007Background: Treatment options for older patients (pts) with acute myelogenous leukemia (AML) unfit for intensive chemotherapy are limited. Expected complete remission rates for low-dose cytarabine (LDAC) are about 10% in this population. Targeting the pro-survival molecule BCL-2 has demonstrated clinical efficacy as a therapeutic strategy in various hematologic malignancies. Venetoclax (VEN), a selective BCL-2 inhibitor, shows synergy with cytarabine in several AML cell lines and primary samples. Methods: This is a non-randomized, open-label phase 1/2 dose-escalation/expansion study of VEN + LDAC, in treatment-naive AML pts ≥ 65 years not eligible for intensive chemotherapy. Pts receive oral VEN once daily (QD) on days 1‒28 and subcutaneous LDAC 20 mg/m2 QD on days 1‒10 of each 28-day cycle. VEN dose escalation follows a 3 + 3 design; dose-limiting toxicities (DLTs: grade 4 toxicity, platelet count < 25,000/μL, or absolute neutrophil count < 500/μL within 14 days of last VEN dose) areassessed during c...

Selective Toxicity of Investigational Ixazomib for Human Leukemia Cells Expressing Mutant Cytoplasmic NPM1: Role of Reactive Oxygen Species.

This study was performed to determine whether the investigational proteasome inhibitor ixazomib demonstrated selective antineoplastic activity against acute myelogenous leukemia cells expressing a mutated nucleophosmin-1 gene and to gain a better understanding of its mechanisms of action. The cytotoxic effects of ixazomib treatment were analyzed in human acute myelogenous leukemia (AML) cell lines and primary AML samples expressing wild-type or mutated NPM1 (NPMc(+)). The potential roles of oxidative stress in mediating cytotoxic activity were determined using flow cytometry, enzyme-based assays, and Western blots. Apoptosis induced by ixazomib was abrogated by knockdown of NPM1/NPMc(+)expression using an inducible shRNA construct and enhanced by NPMc(+)overexpression. Cytotoxicity was associated with superoxide generation and was reduced by the addition of the antioxidant N-acetylcysteine. AML cells expressing NPMc(+)had significantly reduced levels of intracellular glutathione and NADPH associated with reduced antioxidant responses to drug treatment. Treatment of 3 patients with relapsed NPMc(+)AML resulted in an antileukemic effect in 1 patient as demonstrated by a marked reduction of leukemic blasts in the peripheral blood. Efficacy was associated with superoxide generation, reduced glutathione levels, and reduced mRNA and protein expression of antioxidant effectors in responding cells. In this study, a direct association was observed between NPMc(+)expression in AML, reduced antioxidant responses, and enhanced sensitivity to an oral proteasome inhibitor that induces oxidative stress. These data suggest that intracellular determinants of antioxidant responses may be good predictors of therapeutic response to ixazomib.

Optimizing Effects of mTOR Inhibition in Acute Myelogenous Leukemia

Introduction: Mammalian target of rapamycin (mTOR) signaling has previously been identified as a possible therapeutic target in acute myelogenous leukemia (AML), as the PI3K/AKT/mTOR pathway has been shown to be upregulated in blasts of up to 90% of AML cases through the constitutive loss of the tumor suppressor PTEN. The activation of this pathway is implicated in synthesis of prosurvival transcription and translational factors responsible for cellular resistance to apoptosis, resistance to chemotherapy, and enhanced survival in the marrow microenvironment. Unfortunately, exploitation of this pathway has been largely ineffective in clinical studies. A second mTOR complex (mTORC2) will reestablish the activation of the first when the first mTOR complex (mTORC1) is shut down under treatment with traditional rapalogs, such as rapamycin and everolimus. Additionally, the cellular process of autophagy has been postulated to be a protective mechanism for leukemic blasts during treatment with mTOR inhibitors, therefore decreasing their efficacy. For these reasons, this work explores means to optimize mTOR pathway inhibition by examining effects of dual mTORC inhibition (OSI027), dual PI3K/mTOR inhibition (BEZ235/Dactilosib), combination of mTOR inhibitors with a histone deacetylase inhibitor with ability to inhibit AKT (LBH589/Panobinostat), and the combination of mTOR inhibitors with hypomethylating agents able to overcome the hypermethylation of mTOR pathway components such as TSC1, TSC2 and PTEN (decitabine/5-azacytidine).Materials and Methods: Primary AML leukapheresis samples obtained with informed consent were cultured for 48 hours in the presence of OSI027, LBH589 (Novartis), BEZ235 (Novartis), decitabine, or OSI027 in combination with each of these individual inhibitors and compared with control culture conditions. Cells were stained with annexinV and DAPI as previously described, and the percent of living, pre-apoptotic, necrotic and dead cells were determined via flow cytometry. Synergy calculations were completed using Calcusyn™ software as described by Chao and Talalay (1977). Autophagic flux was determined in the OSI027 and LBH589 combination exposure via flow cytometry using anti-LC3. Lastly, protein expression under treatment with various combinations was determined via standard western blotting techniques.Results: The MV411 AML cell line was utilized to establish efficacy of combination exposures. These cells expressed phosphorylated (p)Akt (ser 473), p4EBP1, and pmTOR. OSI027 10µM, LBH589 1µM, and BEZ235 10nM were able to inhibit pmTOR, pAKT, and p4EBP1 expression as determined by western blotting. In the MV411 cell line, IC50s were determined, and combination indices were determined for OSI027+BEZ235, panobinostat+BEZ235, and panobinostat+OSI027; all of which demonstrated synergy. In primary AML samples, the combination of OSI027 10uM and LBH589 10nM significantly increased the percentage of dead cells in comparison to OSI027 treatment alone. Additionally in primary AML blasts, pmTOR and pAKT expression were decreased in the combination of OSI027 and LBH589 when compared to single agent treatment via western blot. The combination of OSI027 and LBH589 demonstrated a significant reduction in the autophagic flux in comparison to OSI027 treatment alone, suggesting an anti-autophagic effect, which correlates with the increased rates of cellular death in the combination. It was also found in AML cell lines that the combination of 10 µM azacytidine and 100 nM rapamycin resulted in synergistic suppression of U937, MV411, and KG1a survival as measured by MTT. No synergy was noted in the HL60 cell line. This combination effectively suppressed CFU-L in primary AML cases and suppressed p70S6K and p4EBP1 expression more than either agent alone by western blotting. The combination of decitabine 500nM with OSI027 10uM did not demonstrate an increase in apoptotic cells in primary blasts compared to OSI027 alone.Conclusions: Based on these findings, we conclude that mTOR inhibition in AML cell lines and primary samples can be enhanced through dual mTORC inhibition, PI3K modulation, and histone deacetylase inhibition. This may occur through suppression of activated pathway mediators or through suppression of autophagic flux. These results suggest that there is merit in exploring these combinations for therapeutic potential in AML. DisclosuresPortwood:ImmunoGen: Research Funding. Wang:ImmunoGen: Research Funding.

Heatshock Protein (HSP) Family Expression Is Dichotomous and Prognostic in Acute Myelogenous Leukemia (AML)

Background. Heat shock family proteins (HSP) are induced by cellular stress, and act as molecular chaperones regulating protein folding, conformation and proteosomal degradation, thereby affecting cell stability, cycling, and apoptosis. High levels of some HSP have been observed in AML and high HSP90 is prognostically adverse. Cell line studies show that HSP protects AML cells against chemotherapy or other stresses and that inhibition of HSP 27, 70 or 90 has antileukemic activity. HSP proteins have typically been studied individually, and in small series. Interactions with multiple signaling and functional pathways are suspected, but the actual relationship in primary AML samples is unknown. We therefore investigated the protein expression of members of the HSPA, B and C groups, HSP 70, 27 and 90, Hugo Names HSPA1A_L, HSPB1 and HSP90AA1_B1, respectively, in a large series of 511 AML patients and compared expression to 228 other simultaneously measured proteins.Methods. We made a reverse phase protein array (RPPA) with protein from leukemia enriched cells from 511 new AML patients. Both bone marrow (BM, n=387) and peripheral blood (PB, n=283) samples were used, with 140 cases having both. The RPPA was probed with 231 strictly validated antibodies, including antibodies against HSP27, 70 and 90. Expression was compared to that of normal bone marrow derived CD34+ cells. Interaction networks with the other 228 proteins were generated from the RPPA data using glasso and supplemented by the literature of known interactions.Results: A heatmap of HSP27, 70 and 90 expression was generated and hierarchical k-and means clustering performed (Fig 1A). Using the “Prototype Clustering” method an optimal division into two clusters C1) characterized by High HSP27 and 70 but low HSP90 and C2) characterized by High HSP90 and low HSP27 and 70 expression. Patients with high HSP90 had significantly higher % BM Blasts (63.6% vs. 42%), WBC (40.8K vs 16.9K), %PB blasts (44 vs. 17%), LDH levels, concurrent infection 34% vs. 12%), FLT3-ITD (25 vs. 13%) or FLT3-D835 mutation (8% vs. 4%) (all P < 0.00001) compared to those with High HSP27 & 70. HSP90 High patients were less likely to have an antecedent hematological disorder (26 vs. 47%, P < 0.00001). Other clinical features, age, gender, cytogenetics, RAS mutation, NPM1 mutation did not differ. Overall survival for all patients (Fig D) was inferior for those with High HSP90 (42 vs. 60 weeks, p = 0.04), but this was based solely on those with intermediate cytogenetics (Fig E) (48 vs. 91 weeks p =< 0.00001), whether FLT-3 mutant (Fig G) (30 vs. 78 weeks, p=0.025) or Wildtype (Fig H, p = 0.037). This was seen in NPM1-WT/FLT3- WT (p=0.03), NPM1-Mut/FLT3 -Mut (fig I, p=0.006) and NPM1-Mut/FLT3 WT (fig J, p=0.08). High HSP90 patients also had inferior remission duration (Fig F, 39 vs. 84 weeks, p=0.025). Networks (Fig B & C) of proteins with expression strongly (p <.00001) correlated with the HSP in the literature (dashed spoke) or the dataset (solid spoke) are shown. Differential expression between the two groups are shown in Fig C (*). Among those with differential expression, levels were higher in the HSP90 high group for histone methylation associated proteins ASH2L, SIRT1, hnRNPK, Nucleolin, proliferation regulators Myc, EIF2S1, EIF2AK2, EIF4E, and for apoptosis/autophagy proteins Beclin1, Park7, MDM2 and BCL2. In contrast protein levels were lower in HSP90 High for : AKT1.pT308, BIRC2, CDKN1A, CDKN2A, CDK4, EGFR 7 pEGFR, ERBB2, HIF1α, IGF1R, LCK PKCα, SRC & pSRC.Conclusions. AML is characterized by dichotomous expression of HSP proteins 27, 70 and 90. HSP expression pattern is associated with FLT3 mutation but not cytogenetics. HSP90 expression is prognostically adverse, exclusively among those with intermediate cytogenetics. High HSP90 expression is associated with higher WBC and PB and BM blast percentages, suggesting a proliferative advantage. This may arise through the effects of Myc and through increased gene expression arising from increased histone3K4 trimethylation from ASH2L and decreased histone 3 acetylation due to increased Sirt1 levels. Therapeutics directed at interfering with HSP function will need to consider which HSP pattern a patient exhibits to target the correct family members. [Display omitted] DisclosuresBorthakur:Tetralogic Pharmaceuticals: Research Funding.

Abstract 4865: Differential survival and self-renewal of LSC and HSC in aged and leukemic niches

Abstract Background: Acute myelogenous leukemia (AML) is a highly lethal disease with only 20% of 5 years survival. Progression to therapy resistant AML is driven by leukemia stem cells (LSC) harboring enhanced survival, dormancy and self-renewal capacity in supportive niches. Evidence indicates that in the development of AML deregulation of stem cells activity is as important as deregulation of the microenvironment. We have recently reported that inflammatory changes in the aging bone marrow (BM) niche via aberrant RNA editing and splicing may predispose patients to leukemic transformation. However, little is known about the functional effects of niche driven aberrant RNA processing in the AML pathogenesis, as well as of role of aberrant RNA editing and splicing in human aged stem cell fate, dormancy and regeneration in the context of microenvironment. Here we investigate: 1/ the LSC impacts to the BM niche and 2/ changes in the activity of BM niche cells contribute to AML pathogenesis. Methods: BM CD34- cell from normal donors and AML patients were used for the development of the primary human stromal monolayers : young (y-BM, n=3); old (a-BM, n=3) and AML (n=3). Human CD34+ cells were selected from AML primary samples (n=6). As a control, CD34+ cells from cord blood (CB, n=5), y-BM (n=3) or a-BM (n=3) were used for the co-culture experiments and then plated in survival and self-renewal assays. Results: AML- and normal BM-derived stroma differ in their ability to support HSC and LSC: LSC (n=6) were capable to self-renew after 9 weeks of co-culture with both normal and AML stroma, while cord blood HSC (n=5) lost their self-renewal potential after only 2 weeks of co-culture with the AML stroma. Aged BM also impaired survival and self-renewal of cord blood HSC (n=3) in stromal co-culture models. In similar experiments HSC from a-BM (n=3) demonstrated significantly higher survival and self-renewal capacity then co-cultured with y-BM stroma (n=3) compared to a-BM stroma (n=3). Conditioned media (CM) both from-, or co-culture with-, old or AML stroma impaired HSC survival and self-renewal. Notably, co-culture conditions resulted in a greater reduction in survival and self- renewal capacity, suggesting that cell-cell contact or unstable secreted factors exacerbate the effects. Cord blood HSC were also co-cultured with AML-derived stroma or CM , and this severely impaired their survival and self-renewal. Moreover, pre-treatment of the HS-5 cells with CM from a-BM or AML stroma for 4 weeks prior to co-culture experiments led to significant inhibition of the cord blood HSC (n=3) survival and self-renewal. Conclusions: Together these data indicate that microenvironmental cues play a key role in regulating normal HSC versus LSC survival and maintenance, and leukemic and aged stroma exibit severely compromised ability to maintain normal HSCs, but effectively support LSCs. Targeting this pathological interplay could represent a novel avenue for treatment of AML. Citation Format: Larisa Balaian, Leslie Crews, Anna Kulidjian, Edward Ball, Catriona Jamieson. Differential survival and self-renewal of LSC and HSC in aged and leukemic niches. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4865. doi:10.1158/1538-7445.AM2014-4865

Immediate utility of two approved agents to target both the metabolic mevalonate pathway and its restorative feedback loop.

New therapies are urgently needed for hematologic malignancies, especially in patients with relapsed acute myelogenous leukemia (AML) and multiple myeloma. We and others have previously shown that FDA-approved statins, which are used to control hypercholesterolemia and target the mevalonate pathway (MVA), can trigger tumor-selective apoptosis. Our goal was to identify other FDA-approved drugs that synergize with statins to further enhance the anticancer activity of statins in vivo. Using a screen composed of other FDA approved drugs, we identified dipyridamole, used for the prevention of cerebral ischemia, as a potentiator of statin anticancer activity. The statin-dipyridamole combination was synergistic and induced apoptosis in multiple myeloma and AML cell lines and primary patient samples, whereas normal peripheral blood mononuclear cells were not affected. This novel combination also decreased tumor growth in vivo. Statins block HMG-CoA reductase (HMGCR), the rate-limiting enzyme of the MVA pathway. Dipyridamole blunted the feedback response, which upregulates HMGCR and HMG-CoA synthase 1 (HMGCS1) following statin treatment. We further show that dipyridamole inhibited the cleavage of the transcription factor required for this feedback regulation, sterol regulatory element-binding transcription factor 2 (SREBF2, SREBP2). Simultaneously targeting the MVA pathway and its restorative feedback loop is preclinically effective against hematologic malignancies. This work provides strong evidence for the immediate evaluation of this novel combination of FDA-approved drugs in clinical trials.

Evaluation of apoptosis induction by concomitant inhibition of MEK, mTOR, and Bcl-2 in human acute myelogenous leukemia cells.

Aberrant activation of multiple signaling pathways is common in acute myelogenous leukemia (AML) cells, which can be linked to a poor prognosis for patients with this disease. Previous research with mTOR or MEK inhibitors revealed cytostatic, rather than cytotoxic, effects in in vitro and in vivo AML models. We evaluated the combination effect of the mTOR inhibitor AZD8055 and the MEK inhibitor selumetinib on human AML cell lines and primary AML samples. This combination demonstrated synergistic proapoptotic effects in AML cells with high basal activation of MEK and mTOR. We next incorporated the BH3 mimetic ABT-737 into this combination regimen to block Bcl-2, which further enhanced the apoptogenic effect of MEK/mTOR inhibition. The combination treatment also had a striking proapoptotic effect in CD33(+)/CD34(+) AML progenitor cells from primary AML samples with NRAS mutations. Mechanistically, upregulation of the proapoptotic protein Bim, accompanied by the downregulation of the antiapoptotic protein Mcl-1 (mainly via protein degradation), seemed to play critical roles in enhancing the combination drug effect. Furthermore, the modulation of survivin, Bax, Puma, and X-chromosome-linked inhibitor of apoptosis protein (XIAP) expression suggested a role for mitochondria-mediated apoptosis in the cytotoxicity of the drug combination. Consequently, the concomitant blockade of prosurvival MEK/mTOR signaling and the deactivation of Bcl-2 could provide a mechanism-based integrated therapeutic strategy for the eradication of AML cells.

Eltrombopag can overcome the anti-megakaryopoietic effects of lenalidomide without increasing proliferation of the malignant myelodysplastic syndrome/acute myelogenous leukemia clone

Lenalidomide (Len) is clinically indicated in myelodysplastic syndrome (MDS) but its use is limited by significant thrombocytopenia. Eltrombopag (EP) is a thrombopoietin receptor agonist that can stimulate platelet production and has shown preclinical efficacy in inhibiting leukemic cell proliferation. Thus, we determined the preclinical efficacy and safety of combining Len and EP in acute myelogenous leukemia (AML) and MDS. We found that single agent treatment of leukemia and lymphoma cell lines with EP and Len showed differential sensitivities to either agent. Combination therapy did not result in reversal of anti-malignant effects on these cells. Furthermore, the combination of Len and EP resulted in significant inhibitory effects on growth of leukemic colonies in the majority of primary MDS and AML samples. Most importantly, EP was able to reverse the anti-megakaryopoietic effects of Len in primary MDS patient samples. These results provide a preclinical rationale for the use of this combination in MDS and AML.

WTAP is a novel oncogenic protein in acute myeloid leukemia

WTAP is a novel oncogenic protein in acute myeloid leukemia

A-Type Proanthocyanidins Prevent Engraftment Of Primary Acute Myelogenous Leukemia Cells In Mice and Exhibit Potentially Novel Anti-Leukemia Mechanisms

Acute myelogenous leukemia (AML) is often a fatal disease where after strong induction therapy most patients relapse and die. AML originates and is maintained by leukemia stem cells (LSCs). Failure to eliminate LSCs by chemotherapy is likely to result in disease relapse. Therefore, it is a priority to identify new therapies that eliminate blasts while ablating LSCs and preventing a relapse.We have found that a unique class of compounds in cranberries (Vaccinium macrocarponAit.), known as A-type proanthocyanidins (A-PACs), were effective against several leukemia cell lines and primary AML samples in vitro. A-PACs consist of monomeric epicatechin units attached to one another by a carbon-carbon bond and a distinctive ether bond that differentiates these compounds from other proanthocyanidins found in nature. Moreover, A-PACs possess ortho-hydroxyl phenolic groups that have the potential to bind to iron and alter redox status.Preliminary work showed that pre-treatment with antioxidants or holo-transferrin (iron-saturated transferrin) partially protected AML cells from A-PAC induced cell death (p<0.01). A-PACs were also found to selectively ablate leukemia stem and progenitor cells, with minimal effects on normal hematopoetic stem cells. Furthermore, AML engraftment of cells treated ex vivo with 62.5 µg/ml A-PACs was decreased (90.6%, n=3, p<0.001), while normal CD34+ cells retained engraftment capability in immunodeficient mice. It was also found that a fraction of A-PACs of up to 7 degree of polymerization was more effective than individual A-PACs. This information prompted us to investigate the in vivo anti-leukemia effects of A-PACs in xenotransplanted mice with primary AML samples, and to further investigate the mechanisms associated with these compounds.Primary AML cells were injected in sub-lethally irradiated NOD/SCID mice. Four weeks after injections, when human leukemia cells have engrafted, intraperitoneal injections of cytarabine (AraC) at 60 mg/kg were given to the mice for 1 week everyday or A-PACs (100 mg/kg dose every 3 days for A-PACs) and vehicle control (1% DMSO in PBS every 3 days) were injected for 2.5 weeks. Mice were sacrificed and leukemia engraftment evaluated using anti-human CD45 and CD33. Moreover, primary cells treated with A-PACs were assessed for effects on iron metabolism, ROS, and survival pathways either by gene expression analysis, flow cytometry or mass spectrometry.Administration of A-PACs to NOD-SCID mice bearing AML tumors reduced tumor burden. Mice that were treated with the vehicle control had engraftment of AML primary cells equivalent to 16.1% (95% CI: -6.0, 38.37; n=4), whereas the mice treated with the A-PACs and AraC showed a level of engraftment of 4.9% (95% CI: 2, 8; n=5) and 5.8% (95% CI: -1.1, 12.7; n=5), respectively. No significant changes in hemoglobin or weight were found between the different treatment groups. Moreover, qPCR analysis of sensitive leukemia cell lines treated with A-PACs showed changes in gene expression of several iron metabolism genes in sensitive leukemia cell lines (up-regulation of ferritin and transferrin receptors 1 and down-regulation of ferroportin) and several ROS-relevant genes (down-regulation of nuclear factor erythroid-2-related factor 2 and glutamate-cysteine ligase regulatory subunit). Mass spectrometry also confirmed that A-PACs bind iron.The results indicate that A-PACs not only target primary AML cells in vitro but are also effective in vivo. Secondary transplants are also being performed to determine the effects on LSC activity. Some of the anti-leukemia mechanisms under investigation include effects related to iron metabolism, ROS or inhibition of survival pathways. Understanding the unique structure and biological effects of A-PACs may provide novel information about pathways involved in the survival of LSCs and provide crucial information in preparation for clinical trials and/or optimal combination drug therapies. Disclosures:Rivella:Novartis: Consultancy; Bayer: Consultancy; Isis: Consultancy, Research Funding; Merganser: Equity Ownership, Research Funding; Biomarin: Consultancy; Alexion: Consultancy; Imago: Consultancy.

Activation Of Akt Enhances Ribosomal RNA Synthesis In AML Cells Through a Novel Isoform Of TIF-1A and Inhibition Of Filamin A Cleavage

PI3Kinase/Akt signaling is frequently activated in acute myelogenous leukemia (AML) and inhibitors of this pathway have shown some promise in both pre-clinical and early clinical studies. In particular, much attention has been focused on components of the mTOR complex as downstream effectors of Akt activation that promote cell proliferation and survival in AML and other malignancies. We have discovered a direct effect of activated and phosphorylated Akt in increasing ribosomal RNA (rRNA) synthesis in both leukemic cell lines and primary AML cells by a novel pathway that is independent of mTOR. We have defined TIF-90 as a splice variant of TIF-1A, a molecule that tethers RNA Polymerase I to the ribosomal DNA promoter and is required for rRNA synthesis. TIF-90 is localized in the nucleolus and directly co-locates with Pol I in imaging, pull down, and rDNA promoter occupancy studies. Activation and phosphorylation of Akt correlates with high levels of TIF-90 expression and increased TIF-90 occupancy of the rDNA promoter in primary AML cells, while overexpression of constitutively activated Akt (Akt-Myr) increases rRNA synthesis in a TIF-90-dependent fashion in cell lines. Activation of Akt increases the expression of TIF-90 by inhibiting its ubiquitination and proteasomal degradation by the p53 ubiquitin ligase, Mdm2. Furthermore, activated Akt directly inhibits the cleavage of the actin-binding protein, Filamin A, into a 90 kDa fragment that interacts with TIF-90 and prevents it from occupying the rDNA promoter. Expression of the 90 kDa Filamin A cleavage fragment alone is sufficient to inhibit rRNA synthesis by binding to TIF-90. Thus, inhibition of Filamin A cleavage by Akt is associated with increased rRNA synthesis. The Akt inhibitor AZD8055, but not the mTORC1 inhibitor Rapamycin, promotes the cleavage of filamin A, decreasing both TIF-90 occupancy of the rDNA promoter and rRNA synthesis. Examination of 21 primary AML samples reveals an inverse correlation between the phosphorylation of Akt and the cleavage of Filamin A; conversely, AZD8055 inhibits both Akt phosphorylation and rRNA synthesis in these samples while promoting the cleavage of Filamin A. These data demonstrate a novel pathway by which Akt directly enhances rRNA synthesis. This pathway is active in primary AML cells and in cell lines that have high levels of phosphorylated Akt. Inhibition of rRNA synthesis has been shown to result in nucleolar disruption, cell cycle arrest, and ultimately cell death. Our data support an mTOR-independent effect of Akt activation on cell proliferation that would require direct targeting of PI3Kinase or activated Akt to have a major therapeutic effect in AML. Disclosures:No relevant conflicts of interest to declare.